Substituted benzamides

ABSTRACT

Substituted benzamides corresponding to the formula I ##STR1## wherein R 1 , R 2  and R 3  have the meanings given herein, and their use in pharmaceutical compositions. The compounds are particularly useful as immunomodulators.

BACKGROUND OF THE INVENTION

This invention relates to substituted benzamides corresponding to thegeneral formula I ##STR2## and their use in medicaments.

Autoimmune diseases arise as a result of a reaction of the immune systemagainst endogenous structures. In the course of this the normallypresent tolerance towards endogenous tissues is suspended. Antibodies,and in particular T-lymphocytes and monocytes/macrophages, play animportant part in the pathogenesis of the various autoimmune diseases.Activated monocytes/macrophages secrete a multitude of differentinflammatory intermediaries, which are directly or indirectlyresponsible for the breakdown of the tissues affected by the autoimmunedisease. The activation of the monocytes/macrophages is mediated eitherby the interaction with T-lymphocytes or by bacterial products such aslipopolysaccharide (LPS). The activation of monocytes/macrophages and ofgranulocytes induced by various bacterial products is moreovercharacteristic of inflammatory reactions in general.

The importance of the equilibrium between inflammatory (for example,interleukin IL-12) and anti-inflammatory cytokines (for example,interleukin IL-10) for the development and progress of inflammation andautoimmune diseases is clearly documented because of numerousexperiments on animals and initial clinical trials. Thepathophysiological significance of IL-12 is apparent in various animalmodels for diseases such as rheumatoid arthritis, multiple sclerosis,diabetes mellitus, as well as inflammatory diseases of the skin andmucous membranes (Immunol. Today 16/8: 383-387, 1995; J. Immunol. 155:4661-4668, 1995; J. Exp. Med. 182: 1281-1290, 1995; J. Exp. Med. 187/4:537-546, 1998). The respective diseases were triggered by theapplication of IL-12 and an abatement of the course of the disease,extending to a recovery of the animals, was apparent afterneutralisation of endogenous IL-12.

In inflammatory bowel diseases, both in diseased animals and in patientssuffering from Crohn's disease, there is a distinctly increased T-cellreactivity in the inflamed sections of the gut. This T-cell reactivityis characterised by the increased expression of IL-12 and IFN- in thelesions. On the other hand, the immunosuppressive cytokine IL-10 isclearly diminished in the lesions (Immunity 3: 171-174, 1995; J. Exp.Med. 182: 1281-1290, 1995; Eur. J. Immunol. 26: 1156-1163; Eur. J.Immunol. 28: 379-389, 1998). The importance of the immunosuppressivecytokine IL-10 for the development of inflammatory intestinal diseasesis also apparent from the fact that IL-10 knockout mice develop aspontaneous colitis (Immunity 3: 171-174, 1995). The activation of theIFN-y-producing T-cells in the lamina propria of the intestine dependssubstantially on the local formation of IL-12. Antibodies to IL-2abrogate established experimental colitis in mice. The neutralization ofIL-12 by antibodies led to a striking improvement in both the clinicaland histopathological aspects of the disease within a few days. No IFN-γproduction could be detected in in vitro activated T-cells from thelamina propria of mice which received anti-IL-12 treatment (J. Exp. Med.182: 1281-1290).

Application of recombinant IL-10 in humans confirms theanti-inflammatory properties. Following the administration of IL-10 tohealthy subjects, the formation of the inflammatory cytokines TNF-α andIL-1 by monocytes activated ex vivo with LPS is reduced by 65 to 95% (J.Immunol. 154: 5492-5499, 1995). The use of IL-10 in patients sufferingfrom steroid-refractory Crohn's disease resulted in an improvement inthe clinical symptoms (Gastroenterology, 113: 383-389). Recently, thesubcutaneous application of IL-10 to three patients suffering frompsoriasis was also reported. There was a marked improvement in thesymptoms of the disease. Moreover, the formation of IL-12 and TNF aswell as the expression of surface molecules on monocytes were alsodecreased (J. Clin. Invest. 101: 783-794). The use of antibodies againstIL-12 in humans is now imminent.

In summary, it can be stated that a deficiency of IL-10 or an excess ofIL-12 determines the pathophysiology of a multitude of inflammatorydiseases. Attempts to normalize the IL-10/IL-12 balance therefore have agreat therapeutic potential.

SUMMARY OF THE INVENTION

Consequently, the object of this invention was the development of newimmunomodulators which do not lead to a general immunosuppression buteffect a normalization of the IL-10/IL-12 balance.

It has now been found that the requirements placed on the substances tobe developed are met by specific substituted benzamides.

Accordingly, the invention provides substituted benzamides correspondingto formula I ##STR3## wherein R¹ represents a group corresponding to theformula COOR⁴, wherein R⁴ denotes a straight-chain or branched alkylgroup having 1 to 6 carbon atoms, or a group corresponding to theformula CONR⁵ R⁶, wherein R⁵ and R⁶ are identical or different anddenote an alkyl group having 1 to 6 carbon atoms (straight-chain orbranched) or, together with the N atom, denote a pyrrolidine,piperidine, hexamethyleneimine or morpholine ring;

R² denotes chlorine, fluorine, CF₃, an alkyl group having 1 to 3 carbonatoms or hydrogen; and

R³ denotes the hydroxyl group, an alkyl or alkoxy group having 1 to 6carbon atoms (straight-chain or branched and optionally substituted withOH--, an alkoxy group, ester group or an open-chain or cyclic amidegroup having 1 to 6 carbon atoms) or a group CH₂ --NR⁵ R⁶, wherein R⁵and R⁶ are as defined above.

The compounds according to the invention can be in racemic form or inenantiomerically pure form or in the form of salts with pharmaceuticallycompatible salts.

DESCRIPTION OF PREFERRED EMBODIMENTS

Preferred substituted benzamides are those in which the group R¹represents a group corresponding to the formula COOR⁴, wherein R⁴denotes a straight-chain or branched alkyl group having 1 to 6 carbonatoms, or a group corresponding to the formula CONR⁵ R⁶, wherein R⁵ andR⁶ together with the N atom denote a morpholine ring; R2 is H and R³denotes the hydroxyl group or a group CH₂ --NR⁵ R⁶, wherein R¹ and R⁶together with the N atom denote a morpholine ring.

The2-(morpholino-4-carbonyl)-N-(1-morpholin-4-ylmethyl-2,6-dioxopiperidin-3-yl)benzamideand the N-(1-hydroxy-2,6-dioxopiperidin-3-yl) phthalamic acid-methylester, are particularly preferred.

Compounds corresponding to the general formula I can be obtained byfirst of all converting, in known manner, a carboxylic acidcorresponding to formula IIa or IIb ##STR4## into an ester (R¹ =COOR⁴)or into an amide (R¹ =CONR⁵ R⁶). Compounds according to the inventionare thus already obtained starting from the carboxylic acid IIb. Thegroup R³ which does not denote the hydroxyl group can then be introducedinto these compounds derived correspondingly from the carboxylic acidIIa, again in known manner, for example, by Mannich reaction withparaformaldehyde and a secondary amine corresponding to the formula HNR⁵R⁶.

The invention also provides the use of the substituted benzamidescorresponding to formula I as medicaments, in particular asimmunomodulators. The substances according to the invention clearlyinhibit the production of the inflammatory cytokine IL-12 byLPS-activated human monocytes. On the other hand, substances of thisgroup increase the production of the anti-inflammatory cytokine IL-10 byLPS-activated human monocytes. This distinguishes the new substancesfrom known immunomodulators such as steroids and phosphodiesteraseinhibitors, which suppress the synthesis of IL-12 as well as that ofIL-10. Due to their characteristic immunomodulatory activity (inhibitionof IL-12, increase of IL-10), the substances according to the inventionare suitable for the treatment and/or prophylaxis of inflammation, inparticular inflammation of the skin and mucous membranes and of thevessels, as well as for the treatment and/or prophylaxis of autoimmunediseases.

These diseases include, inter alia, inflammation of the skin (forexample, atopic dermatitis, psoriasis, eczema), inflammation of therespiratory tract (for example, bronchitis, pneumonia, bronchial asthma,ARDS (adult respiratory distress syndrome), sarcoidosis,silicosis/fibrosis), inflammation of the gastrointestinal tract (forexample, gastroduodenal ulcer, Crohn's disease, ulcerative colitis),also diseases such as hepatitis, pancreatitis, appendicitis,peritonitis, nephritis, aphthous ulcers, conjunctivitis, keratitis,uveitis, rhinitis.

The autoimmune diseases include, for example, diseases of the arthritictype (for example, rheumatoid arthritis, HLA-B27 associated diseases),also multiple sclerosis, juvenile diabetes or lupus erythematosus.

Further indications are sepsis, bacterial meningitis, cachexia,transplant rejection reactions, graft-versus-host reactions as well asreperfusion syndrome and atherosclerosis.

Medicaments according to the invention contain at least one compoundcorresponding to the general formula I and in addition carriers,fillers, solvents, diluents, dyes and/or binders. The selection of theauxiliary substances and the quantities to be used depend on whether themedicament is to be administered orally, intravenously,intraperitoneally, intradermally, intramuscularly, intranasally,buccally or locally. Suitable preparations for oral administration arein the form of tablets, chewing tablets, dragees, capsules, granules,droplets, juices or syrups. Solutions, suspensions, readilyreconstitutable dry preparations and sprays are suitable for parenteraland topical administration and for inhalation. Compounds according tothe invention in a depot in dissolved form, on a supporting film or aplaster, optionally with the addition of agents which promotepenetration of the skin, are examples of suitable percutaneous forms ofadministration. Delayed release of the compounds according to theinvention from orally or percutaneously administrable forms ofpreparation is also possible.

The quantity of active ingredient to be administered to patients variesdepending on the weight of the patient, on the method of administration,the indication and the severity of the disease. Generally, 1 to 150mg/kg of at least one compound according to the invention correspondingto formula I is administered.

EXAMPLES

                  TABLE 1                                                         ______________________________________                                                   Structure of                                                                  substance                                                          Example No.                                                                              Formula I with  Name                                               ______________________________________                                        1          R.sup.1 =                                                                            COOH         N-(1-morpholin-4-                              (Comparison                                                                              R.sup.2 =                                                                            H            ylmethyl-2,6-                                  Example)   R.sup.3 =                                                                            CH.sub.2 --NR.sup.5 R.sup.6,                                                               dioxopiperidin-3-                                                wherein R.sup.5                                                                            yl)phthalamic acid                                               and R.sup.6                                                                   together with                                                                 the N atom                                                                    denote a                                                                      morpholine ring                                             2          R.sup.1 =                                                                            H            N-(1-morpholin-4-                              (Comparison                                                                              R.sup.2 =                                                                            H            ylmethyl-2,6-                                  Example)   R.sup.3 =                                                                            CH.sub.2 --NR.sup.5 R.sup.6,                                                               dioxopiperidin-3-                                                wherein R.sup.5                                                                            yl)benzamide                                                     and R.sup.6                                                                   together with                                                                 the N atom                                                                    denote a                                                                      morpholine ring                                             3          R.sup.1 =                                                                            CONR.sup.5 R.sup.6,                                                                        2-(morpholine-4-                               (according to     wherein R.sup.5                                                                            carbonyl)-N-(1-                                the               and R.sup.6  morpholin-4-                                   invention)        together with                                                                              ylmethyl-2,6-                                                    the N atom   dioxopiperidin-3-                                                denote a     yl)benzamide                                                     morpholine ring                                                        R.sup.2 =                                                                            H                                                                      R.sup.3 =                                                                            CH.sub.2 --NR.sup.5 R.sup.6,                                                  wherein R.sup.5                                                               and R.sup.6                                                                   together with                                                                 the N atom                                                                    denote a                                                                      morpholine ring                                             4          R.sup.1 =                                                                            COOCH.sub.3  N-(1-hydroxy-2,6-                              (according to                                                                            R.sup.2 =                                                                            H            dioxoperidin-3-                                the        R.sup.3 =                                                                            OH           yl)phthalamic                                  invention)                     acid-methyl ester                              5          R.sup.1 =                                                                            CONR.sup.5 R.sup.6,                                                                        2-(morpholine-4-                               (according to     wherein R.sup.5                                                                            carbonyl)-N-(1-                                the               and R.sup.6  piperidin-4-                                   invention)        together with                                                                              ylmethyl-2,6-                                                    the N atom   dioxopiperidin-3-                                                denote a     yl)benzamide                                                     morpholine ring                                                        R.sup.2 =                                                                            H                                                                      R.sup.3 =                                                                            CH.sub.2 --NR.sup.5 R.sup.6,                                                  wherein R.sup.5                                                               and R.sup.6                                                                   together with                                                                 the N atom                                                                    denote a                                                                      piperidine ring                                             6          R.sup.1 =                                                                            COOC.sub.2 H.sub.5                                                                         N-(2,6-dioxo-1-                                (according to                                                                            R.sup.2 =                                                                            H            piperidin-1-yl-                                the        R.sup.3 =                                                                            CH.sub.2 --NR.sup.5 R.sup.6,                                                               methyl-piperidin-                              invention)        wherein R.sup.5                                                                            3-yl)phthalamic                                                  and R.sup.6  acid-ethyl ester                                                 together with                                                                 the N atom                                                                    denote a                                                                      piperidine ring                                             ______________________________________                                    

The substances in Table 1 were analyzed by ¹ H-NMR spectroscopy(equipment: DPX 300 Avance, from the firm Bruker; 300 MHz; solvent:DMSO-d₆ ; data on chemical shifts in ppm).

Example 1

1.97-2.16 (m, 2H, CH₂); 2.52-2.86 (m, 2H, CH₂) 3.20-3.75 (m, 8H, CH₂);4.50-4.65 (m, 2H, NCH₂ N) 4.64-4.82 (m, 1H, CH); 7.49-7.66 (m, 3H,aromat.); 7.79-7.85 (d, 1H, aromat.); 8.60-8.72 (d, 1H, CONH).

Example 2

1.94-2.20 (m, 2H, CH₂); 2.68-2.95 (m, 2H, CH₂); 3.28-3.70 (m, 8H, CH₂);4.52-4.65 (m, 2H, NCH₂ N); 4.83-4.96 (m, 1H, CH); 7.42-7.58 (m, 3H,aromat.); 7.85-7.92 (m, 2H, aromat.); 8.85-8.89 (d, 1H, CONH).

Example 3

1.92-2.24 (m, 2H, CH₂); 2.69-3.02 (m, 2H, CH₂); 3.12 (s, 2H, CH₂);3.35-3.77 (m, 12H, CH₂) 4.52-4.68 (m, 2H, NCH₂ N); 4.74-4.98 (m, 1H,CH); 7.28-7.32 (d, 1H, aromat.); 7.45-7.59 (m, 2H, aromat.); 7.74-7.80(d, 1H, aromat.); 8.78-8.88 (d, 1H, CONH).

Example 4

2.01-2.18 (m, 2H, CH₂); 2.58-2.88 (m, 2H, CH₂); 3.79 (s, 3H, COOCH₃);4.70-4.80 (m, 1H, CH); 7.54-7.72 (m, 3H, aromat.); 7.77-7.81 (d, 1H,aromat.); 8.77-8.82 (d, 1H, CONH); 10.21 (s, 1H, NOH).

Example 5

1.38-1.46 (m, 6H, CH₂); 1.96-2.16 (m, 2H, CH₂); 2.20-2.32 (m, 4H, CH₂);2.60-2.92 (m, 2H, CH₂); 3.26-3.75 (m, 8H, CH₂); 4.57-4.70 (m, 2H, NCH₂N) 4.72-4.88 (m, 1H, CH); 7.28-7.30 (d, 1H, aromat.); 7.43-7.57 (m, 2H,aromat.); 7.70-7.74 (m, 1H, aromat.); 8.75-8.78 (d, 1H, CONH).

Example 6

1.18-1.26 (t, 3H, CH₃); 1.36-1.48 (m, 6H, CH₂); 1.95-2.16 (m, 2H, CH₂);2.22-2.34 (m, 4H, CH₂); 2.60-2.90 (m, 2H, CH₂); 4.08-4.18 (q, 2H, OCH₂);4.55-4.68 (m, 2H, NCH₂ N); 4.70-4.80 (m, 1H, CH); 7.54-7.72 (m, 3H,aromat.); 7.78-7.83 (d, 1H, aromat.); 8.78-8.82 (d, 1H, CONH).

Investigation of the immunomodulatory activity

Human monocytes were isolated from peripheral blood mononuclear cells(PBMC), which had been obtained from heparinized whole blood by means ofa Ficoll density-gradient centrifugation. To this end, the PBMC wereincubated with a monoclonal antibody which was directed against themonocyte-specific surface molecule CD14 and to which superparamagneticmicrobeads (Miltenyi Biotech, Bergisch Gladbach) were coupled. To effecta positive selection of the labelled monocytes from the cell mixture ofthe PBMC, the total cell suspension was applied to a column withferromagnetic supporting matrix and this was placed in a magnetic field.By this means the cells, which were labelled with microbeads, were boundto the supporting matrix, unlabelled cells passed through the column andwere discarded. After the removal of the matrix out of the magneticfield, the cells labelled with antibodies were eluted by rinsing the nowdemagnetised column with a buffer. The purity of this CD14-positivemonocyte population thus obtained was about 95 to 98%. These monocyteswere incubated--at a density of 10⁶ cells/ml culture medium (RPMI,supplemented with 10% foetal calf serum)--together with the testsubstances dissolved in DMSO, for one hour at 37° C. and 5% CO₂. Then 20μg/ml LPS from E. coli was added thereto. After 24 hours, cell-freeculture supernatants were taken and analyzed to determine the content ofthe cytokines IL-12 and IL-10.

The concentration of IL-12 and IL-10 in the cell culture supernatantswas determined by means of sandwich-ELISAs using two anti-IL-12 andanti-IL-10 monoclonal antibodies (Biosource Europe, Fleurus, Belgium). Astandard reference curve for human IL-12 and IL-10 respectively wasincluded. The detection limit of the IL-12 ELISA was 10 pg/ml and thatof the IL-10 ELISA was 15 pg/ml.

                  TABLE 2                                                         ______________________________________                                        Influence of the test substances on the                                       production of IL-12 and IL-10 by LPS-activated monocytes.                                         IL-12 production                                                                          IL-10 production                                        Concen-   % of the control                                                                          % of the control                              Substance tration   (=100%)     (=100%)                                       ______________________________________                                        from      10 μg/ml                                                                             76          109                                           Example 1 2 μg/ml                                                                              78          84                                                      0.4 μg/ml                                                                            90          87                                                      6.0 μg/ml                                                                            105         69                                            from      2.0 μg/ml                                                                            104         99                                            Example 2 0.66 μg/ml                                                                           102         109                                                     0.22 μg/ml                                                                           99          114                                           from      10 μg/ml                                                                             30          127                                           Example 3 2 μg/ml                                                                              46          121                                                     0.4 μg/ml                                                                            87          108                                           from      6.0 μg/ml                                                                            49          131                                           Example 3 2.0 μg/ml                                                                            53          133                                           (second   0.66 μg/ml                                                                           59          123                                           test)     0.22 μg/ml                                                                           64          117                                           from      10 μg/ml                                                                             23          112                                           Example 4 2 μg/ml                                                                              40          189                                                     0.4 μg/ml                                                                            66          122                                           from      6.0 μg/ml                                                                            47          134                                           Example 5 2.0 μg/ml                                                                            57          134                                                     0.66 μg/ml                                                                           67          122                                                     0.22 μg/ml                                                                           88          115                                           from      6.0 μg/ml                                                                            38          130                                           Example 6 2.0 μg/ml                                                                            47          124                                                     0.66 μg/ml                                                                           61          132                                                     0.22 μg/ml                                                                           79          102                                           Dexa-     1 μM   6           34                                            methasone 0.1 μM 6           35                                                      0.01 μM                                                                              20          64                                                      0.001 μM                                                                             83          105                                           Pentoxi-  50 μg/ml                                                                             74          74                                            fylline   5 μg/ml                                                                              72          81                                            Rolipram  50 μM  32          28                                                      0.5 μM 30          79                                                      0.005 μM                                                                             58          92                                            ______________________________________                                    

The results presented in Table 2 show that known immunomodulators suchas dexamethasone, pentoxifylline and Rolipram suppress both theproduction of IL-12 and that of IL-10 by LPS-activated monocytes. Incomparison, structurally similar benzamides substituted with carboxylgroups (Example 1) exhibit only a slight action at high doses.

Surprisingly, the esters according to the invention (Examples 4 and 6)and the amides according to the invention (Examples 3 and 5) of thesubstituted benzamides exhibit immunomodulatory activity in the modelinvestigated. Such compounds powerfully inhibit the synthesis of IL-12by LPS-activated monocytes at concentrations of 10, 6 and 2 μg/ml,respectively. Unlike the known immunomodulators, they also increase thesynthesis of IL-10. This characteristic pattern (clear inhibition ofIL-12, increase of IL-10 in the same concentration range of substances)distinguishes a new type of immunomodulator.

The foregoing description and examples have been set forth merely toillustrate the invention and are not intended to be limiting. Sincemodifications of the disclosed embodiments incorporating the spirit andsubstance of the invention may occur to persons skilled in the art, theinvention should be construed broadly to include all variations fallingwithin the scope of the appended claims and equivalents thereof.

What is claimed is:
 1. A substituted benzamide corresponding to formulaI ##STR5## wherein R¹ represents a group corresponding to the formulaCOOR⁴, wherein R⁴ denotes a straight-chain or branched alkyl grouphaving 1 to 6 carbon atoms, or a group corresponding to the formulaCONR⁵ R⁶, wherein R⁵ and R⁶ are identical or different and denote astraight-chain or branched alkyl group having 1 to 6 carbon atoms or,together with the N atom, denote a pyrrolidine, piperidine,hexamethyleneimine or morpholine ring;R² denotes chlorine, fluorine,CF₃, an alkyl group having 1 to 3 carbon atoms or hydrogen, and R³denotes the hydroxyl group, a straight-chain or branched alkyl or alkoxygroup having 1 to 6 carbon atoms and optionally substituted with OH--,an alkoxy group, ester group or an open-chain or cyclic amide grouphaving 1 to 6 carbon atoms, or R³ denotes a group CH₂ --NR⁵ R⁶, whereinR⁵ and R⁶ are as defined above.
 2. A substituted benzamide according toclaim 1, whereinR¹ represents a group corresponding to the formulaCOOR⁴, wherein R⁴ denotes a straight-chain or branched alkyl grouphaving 1 to 6 carbon atoms, or a group corresponding to the formulaCONR⁵ R⁶, wherein R⁵ and R⁶ together with the N atom denote a morpholinering; R² is H, and R³ denotes the hydroxyl group or a group CH₂ --NR⁵R⁶, wherein R⁵ and R⁶ together with the N atom denote a morpholine ring.3. A pharmaceutical composition comprising an effective immunomodulatingamount of a substituted benzamide according to claim 1, and at least onepharmaceutical carrier or adjuvant.
 4. A method of treating a patientsuffering from a deficiency of IL-10 or an excess of IL-12, said methodcomprising the step of administering to said patient an effectiveimmunomodulating amount of a substituted benzamide according to claim 1.